A robust metaproteomics pipeline for a holistic taxonomic and functional characterization of microbial communities from marine particles

Abstract

38This study aimed to establish a robust, reproducible and reliable metaproteomic pipeline for 39 an in-depth characterization of marine particle-associated (PA) bacteria. To this end, we 40 compared six well-established protein extraction protocols together with different MS-sample 41 preparation techniques using particles sampled during a North Sea spring algae bloom in 422009. In this optimized workflow, proteins are extracted using a combination of SDS-43 containing lysis buffer and cell disruption by bead-beating, separated by SDS-PAGE, in-gel 44 digested and analysed by LC-MS/MS, before MASCOT search against a metagenome-45 based database and data processing/visualization with the in-house-developed 46 bioinformatics tools Prophane and Paver. 47As proof of principle, free-living (FL) and particulate communities sampled in April 2009 48 were analysed, resulting in an as yet unprecedented number of 9,354 and 5,034 identified 49 protein groups for FL and PA bacteria, respectively. Our data revealed that FL and PA 50 communities appeared similar in their taxonomic distribution, with notable exceptions: 51 eukaryotic proteins and proteins assigned to Flavobacteriia, Cyanobacteria, and some 52 proteobacterial genera were found more abundant on particles, whilst overall proteins 53 belonging to Proteobacteria were more dominant in the FL fraction. In contrast, significant 54 functional differences including proteins involved in polysaccharide degradation, sugar-and 55 phosphorus uptake, adhesion, motility, and stress response were detected. 56 7 Results and Discussion 128Establishment of a metaproteomics pipeline for PA microbial communities 129As stated above, the metaproteomics analysis of PA microbial communities is severely 130 hampered by their high complexity, the presence of a large proportion of eukaryotic proteins, 131 the sugary particle-matrix as well as the lack of (meta)genomic information on PA-specific 132 pro-and eukaryotes (Wöhlbrand et al., 2017b; Saito et al., 2019). Whilst the metaproteomics 133 analyses by Teeing et al. (2012) and Kappelmann et al. (2019) of FL bacterioplankton 134 (harvested on 0.2 µm filters) sampled during spring blooms from 2009 to 2012 at 135 "Kabeltonne" Helgoland resulted in the identification of several thousand protein groups, the 136